Light scattering and absorption model for the quantitative interpretation of human blood platelet spectral data.

Multiwavelength ultraviolet-visible (UV-Vis) transmission spectroscopy is a relatively simple technique that can provide considerable quantitative information on the properties of micron and submicron particle suspensions. Two important particle properties are particle size distribution (PSD) and chemical composition. These properties provide characteristics for the identification and classification of biological systems ranging in size and composition from proteins and nucleic acids to cells. By measuring the complete UV-Vis spectrum, the combined scattering and absorption properties are obtained as a function of wavelength. The quantitative evaluation of the size distribution and chemical composition is accomplished through the application of light-scattering theory. This paper reports on the estimation of the optical properties of human blood platelets and their use in the interpretation of platelet UV-Vis spectra within the context of Mie theory. The model developed herein provides reliable and accurate estimates for the PSD and particle number of platelet suspensions. One potential application of this characterization method is in the analysis of platelet activation by thrombin. Quantification of spectral data with respect to average particle size and particle number provides a real-time description of the dramatic changes that accompany the platelet activation process.

Ultraviolet and visible light spectrophotometric approach to blood typing: objective analysis by agglutination index.

BACKGROUND: A new blood typing technology based on ultraviolet (UV) and visible light spectroscopy (UV/visible spectroscopy) has been developed. Blood groups and types are determined by quantifying reproducible changes in the UV and visible light spectra of blood in the presence of agglutinating antibodies. STUDY DESIGN AND METHODS: Samples of red cells in the presence and absence of agglutinating antibodies were examined by UV/visible spectroscopy. Blood groups and types were determined by comparing the optical density spectra obtained between 665 and 1000 nm. These comparisons generate numbers (agglutination index) ranging from 0 to 100, with smaller numbers corresponding to lack of agglutination and larger numbers corresponding to agglutination. RESULTS: The optical density of agglutinated blood is dramatically different from that of unagglutinated blood. The agglutination index derived from the relative slopes of the spectra is an objective indicator of agglutination strength. An agglutination index greater than 17 consistently and accurately established blood group- and type-specific agglutination. CONCLUSION: The method accurately predicted A, B, and O blood groups, and D type in over 275 samples. Scattering theory-based calculations of relative volumes of red cells before and after agglutination show a direct correlation with the agglutination index and provide the theoretical basis of the analysis. This quantitative technique is reproducible and has the potential for automation.

Cytotoxicity of paraquat in freshly isolated rat hepatocytes: effects of L-carnitine.

The effects of L-carnitine, a mitochondrial carrier of fatty acids, on paraquat (PQ) cytotoxicity in freshly isolated rat hepatocytes were studied. Addition of PQ (10 mM) to hepatocytes resulted in a time-dependent depletion of intracellular glutathione (GSH) accompanied by an increase in accumulation of malondialdehyde (MDA) in the incubation medium which proceeded to a loss of cell viability. Pretreatment of hepatocytes with L-carnitine (50-mM) alone did not affect cell viability or intracellular levels of GSH, or accumulation of MDA in the medium during the incubation period; however, pretreatment with L-carnitine 30 min prior to PQ addition did promote the depletion of intracellular GSH and MDA accumulation induced by PQ, and ultimately enhanced the cytotoxicity of PQ.

Accentuation of paraquat-induced toxicity by L-carnitine in mice.

The effects of L-carnitine on toxicity induced by paraquat (PQ) in mice were investigated. L-carnitine pretreatment surprisingly promoted the toxicity of PQ in a dose-dependent manner shortening the survival time. The maximum effect occurred when L-carnitine, at a dose of 16 mmol/kg, was intraperitoneally administered 30 min before an intraperitoneal injection of PQ (75 mg/kg). Lipid peroxidation in lung significantly increased 6 h after PQ administration. L-carnitine accelerated this effect since L-carnitine-pretreated mice already showed a significant increase of lung malondialdehyde 1 h after PQ administration. In liver, PQ administration did not produce lipid peroxidation; nevertheless L-carnitine-pretreated mice showed a significant increase of malondialdehyde 6 h after PQ administration. Lung and liver glutathione decreased in mice receiving only PQ but this effect was not significantly changed by L-carnitine pretreatment. These results indicate that L-carnitine accelerates PQ-induced mortality rate by facilitating lipid peroxidation.

A physicochemical comparison of the isozymes of creatine kinase from rabbit brain and muscle.

A comparison of specific structural features of creatine kinase from rabbit muscle and brain was undertaken to determine if the observed isozyme specific differences in catalytic cooperativity are related to conformational differences, particularly differences in packing density. The intrinsic fluorescence of the brain isozyme is 2-fold higher than the muscle isozyme. In the denatured state, both proteins display the characteristic red shift in emission maximum; however, the emission intensity of the brain isozyme increases only 5% upon denaturation compared to nearly 100% increase for the muscle protein. The fluorescence lifetimes are 2.65 ns (67%) and 0.48 ns for native muscle enzyme and 4.38 ns (65%) and 0.80 ns for brain enzyme. Upon denaturation, the lifetimes are 3.98 ns (77%) and 0.99 ns for muscle protein and 3.82 ns (79%) and 0.86 ns for brain protein. Stern-Volmer plots of quenching by acrylamide are essentially the same for both native isozymes indicating that the differences of the intrinsic fluorescence of the native proteins are not due to differences in solvent accessibility. The spectral and lifetime differences in the isozymes in the native state and changes accompanying denaturation are consistent with the occurrence of energy transfer in native muscle isozyme. The rotational correlation times of 5-[2-(iodoacetyl)aminoethyl]aminonaphthalene-1-sulfonate conjugated proteins, derivatized at the active site reactive thiol, are best described by two term decay laws. The slower rotations, 45.1 ns (75%) and 40.6 ns (71%) reflect overall macromolecular rotation for the muscle and brain isozymes, respectively. The faster motions, 2.4 ns for muscle isozyme and 0.4 ns for the brain isozyme, are attributed to the probe or probe associated segmental motions and indicate these motions are more restricted in the muscle protein. Reactivity of creatine kinase (2.5-10 microM) with the amino-specific reagent trinitrobenzene sulfonate (0.4-2 mM) was analyzed by pseudo-first-order and second order models, neither of which was adequate for the entire range of data. However, in every case, the rate constants were faster for brain creatine kinase but the extent of reaction was greater for muscle creatine kinase. The faster initial reactivity of the brain isozyme is consistent with greater accessibility for lysine derivatization.(ABSTRACT TRUNCATED AT 400 WORDS)

Interaction of brain-type creatine kinase with its transition state analog: kinetics of inhibition and conformational changes.

The effects of components of the transition state analog (creatine, MgADP, planar anion) on the kinetics and conformation of creatine kinase isozyme BB from monkey brain was studied. From analysis of the reaction time course using the pH stat assay, it was shown that during accumulation of the reaction products (ADP and creatine phosphate), among several anions added, nitrate proved the most effective in inhibiting catalytic activity. Maximum inhibition (77%) was achieved with 50 mM nitrate. The Km for ATP was 0.48 mM and in the presence of 2.5 mM nitrate, 2.2 mM; for ATP in the presence of the dead-end complex, creatine and ADP, the apparent Km was 2.0 mM and the Ki was 0.16 mM; in the presence of the transition state analog, MgADP + NO3- + creatine, the Ki was estimated to be 0.04 mM. Ultraviolet difference spectra of creatine kinase revealed significant differences only in the presence of the complete mixture of the components of the transition state analog. Comparison of gel filtration elution profiles for creatine kinase in the absence and presence of the complete mixture of components of the transition state analog did not reveal any differences in elution volume. Addition of components of the transition state analog to creatine kinase resulted in only a marginal change in intrinsic fluorescence. The presence of the components of the transition state analog increased the rate of reactivity of the enzyme with trinitrobenzenesulfonic acid from k = 6.06 +/- 0.05 M-1 min-1 to 6.96 +/- 0.11 M-1 min-1.(ABSTRACT TRUNCATED AT 250 WORDS)